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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology and Physiology of Biting Flies Affecting Livestock

Location: Tick and Biting Fly Research

2013 Annual Report


1a.Objectives (from AD-416):
Objective 1. Explore the genetic and physiological mechanisms of stable fly feeding and reproduction to identify novel control targets and to develop more efficient behavior modifying compounds. Sub-objective 1.A. Identify and characterize genes that have a role in the olfactory and gustatory pathways of biting flies. Sub-objective 1.B. Elucidate the mechanisms of blood-feeding in biting flies by characterizing the structure and neurophysiology of the cibarial pump, a key component of the feeding system for blood ingestion in the stable fly and other blood-feeding fly species. Sub-objective 1.C. Identify key neurotransmitters and/or receptors from biting flies and characterize their roles in mating and egg-laying behaviors. Objective 2. Develop gene silencing tools to facilitate the functional characterization of novel control targets in biting flies, with a particular emphasis on genes that play a role in feeding and reproduction. Objective 3. Develop genomic resources to support the initiation of a genome sequencing project for biting flies that impact livestock.


1b.Approach (from AD-416):
The objectives of this project will be achieved using multidisciplinary approaches including molecular biology, immunohistochemistry, neurophysiology, and behavioral assays. Genes that play a critical role in olfaction and gestation of biting flies will be identified and characterized using pyrosequencing technology. Messenger RNA will be isolated from dissected olfactory and gustatory organs of the stable fly and used as template in the synthesis of double-stranded cDNA. Annotation of the stable fly transcriptome database representing genes expressed at different developmental stages will be accomplished by comparison to Drosophila sequences. Sequences encoding putative chemoreceptors will be isolated. The temporal and spatial expression patterns of the chemosensory gene sequences will be characterized using non-quantitative reverse transcriptase PCR and in situ hybridization techniques. The mechanisms of blood feeding in biting flies will be determined by identifying neurotransmitters in the feeding system and characterizing the cibarial pump function. Immonohistological techniques will be used to localize the specific neurotransmitters in neurons innervating the cibarial muscles. An in vitro blood feeding system will be developed and used in conjunction with the electrophysiological recording system to characterize cibarial pump function. Neurotransmitters (receptors) that are critical for blood feeding will be determined through pharmacological experiments involving agonists and antagonists. Neurotransmitters (receptors) that are critical to biting fly reproduction will be similarly identified and characterized. Immunohistological techniques will be used to identify specific neurotransmitters in neurons innervating testes in males and ovary/oviduct in females. Roles of specific neurotransmitters (receptors) in sperm transfer and egg-laying will be determined through behavioral and pharmacological experiments. Neurotransmitters that are critical for egg-laying behaviors will be further characterized by electrophysiological recordings of oviduct contraction in reduced fly preparations. Genes encoding receptors of key neurotransmitters in the sensory, feeding and reproductive systems will be identified. Gene-silencing tools will be developed to facilitate the functional characterization of novel control targets, particularly on genes that play critical roles in blood feeding and reproduction of biting flies. The double-stranded RNA (dsRNA) of a target gene will be synthesized and used for gene silencing. Microinjection techniques that are suitable for injecting dsRNA will be adopted from available insect protocols and be optimized for injecting the stable fly. The effects of gene silencing will be evaluated by measurement of transcript reduction using quantitative real-time PCR and/or by monitoring changes in key behaviors, including responses to chemical cues and mating /egg-laying success. Finally, a first generation genetic linkage map will be developed and a bacteria artificial chromosome (BAC) library will be constructed to support the initiation of a genome sequencing project for biting flies affecting livestock.


3.Progress Report:
As part of our continued interest in evaluating insecticides for biting fly control, our lab completed assessment of a novel benzoylphenylurea against horn, house, and stable flies. The tested insecticide had an IGR-like impact on the developing fly larvae, causing visible pupal deformity and significant reduction of adult fly emergence. Further, our lab has made progress in understanding the horn fly's molecular response to insecticide applications. A quantitative genomics study of pesticide-treated and -untreated flies identified the suite of enzymes comprising the horn fly metabolome. The genes that encode these enzymes respond to various xenobiotics, including pesticides, and individual members of the metabolome have been found that likely provide metabolic resistance to pyrethroids and organophosphates. To understand the effect of biogenic amines on biting fly feeding activity, our lab was able to make direct recordings of the stable fly cibarial pump muscle and identify rhythmic activity and distinct patterns that correlate with blood ingestion and salivation. These recorded patterns were affected by exposure to several pharmacological agents, including pilocarpine, serotonin, dopamine, octopamine, and tyramine. In an effort to identify insecticidal chemistries that are species-selective and display low levels of toxicity to the mammalian host, we collaborated with university scientists to evaluate a synthetic carbamate PRC-408 for inhibition of recombinant acetylcholinesterases (rAChEs) of horn, stable, and sand flies. PRC-408 is believed to offer improved safety compared to other AChE inhibitors, as it exhibited approximately 300-fold higher specificity for arthropod AChEs compared to mammalian AChEs (bovine and human). In addition, PRC-408 was evaluated by in vivo bioassay against horn, stable, and sand flies, and it exhibited insecticidal activity comparable to that of carbaryl (an efficacious insecticide). Recently, efforts have been made by our lab to evaluate the effect of systemic parasiticides on endemic populations of Aedes albopictus and Culex quinquefasciatus. To this end, we have successfully fed citrated bovine blood to both of these mosquito species via an in vitro system using stretched parafilm as a membrane; this system will be used in parasiticide evaluation. The insect-microbe relationship is critical to biting fly biology. As a result, we evaluated the interaction of specific bacterial species with either the stable or horn fly upon ingestion by adults, and these studies indicated that specific bacteria persist differently in the two fly species. In parallel, we initiated studies to identify genes that may have a role in enabling biting fly survival in microbe-rich environments, i.e., innate immune system genes, and we plan to investigate the role these molecules may have in explaining the differences in bacteria persistence within biting flies. In support of a stable fly genome sequencing project, we provided high quality genomic DNA from our lab-derived genome sequencing strain to a collaborating genome institute, and the sample is currently in the sequencing queue.


4.Accomplishments
1. Characterization of a synthetic carbamate insecticide with low toxicity to mammalian hosts. Attaining species-selectivity can at times be a hurdle to the development of new or modified insecticidal chemistries. ARS scientists at Kerrville, Texas, collaborated with researchers at the University of Florida and Virginia Tech to evaluate a synthetic carbamate insecticide, PRC-408, for the control of horn, stable, and sand flies. PRC-408 demonstrated insecticidal activity comparable to that of carbaryl, an efficacious, commercially available compound. Most importantly, an in vitro assay was used to demonstrate that PRC-408 exhibited approximately 300-fold higher specificity for its arthropod target compared to its mammalian, i.e., bovine and human, target and offers improved safety compared to other chemicals in its class.

2. Assembly of a comprehensive dataset of genes expressed by the horn fly. Current paradigms in medical and pharmaceuticals research and development are genome-centric. Genome and transcriptome datasets are lacking for biting flies, and this hampers the development of novel control technologies. ARS scientists at Kerrville, Texas, in collaboration with scientists at Louisiana State University and the National Center for Genome Resources at Santa Fe, New Mexico, have synthesized, sequenced, and annotated a comprehensive transcriptome of the horn fly, representing genes expressed during the embryonic, larval, and adult stages of the fly. This dataset will serve as the foundation of a genome sequencing project for the horn fly and as a valuable resource for studies aimed at development of anti-fly vaccines, identification of novel targets for new pesticides, or production of transgenic flies for genetic control programs.


Review Publications
Olafson, P.U. 2013. Molecular characterization and immunolocalization of the olfactory co-recepter orco from two blood-feeding muscid flies, the stable fly (Stomoxys calcitrans, L.) and the horn fly (Haematobia irritans irritans, L.). Insect Molecular Biology. 22(2):131-142.

Swale, D.R., Tong, F., Temeyer, K.B., Li, A.Y., Lam, P., Totrov, M.M., Carlier, P.R., Perez De Leon, A.A., Bloomquist, J.R. 2013. Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi. Journal of Pesticide Biochemistry and Physiology. 106:85-92.

Temeyer, K.B., Brake, D.K., Tuckow, A.P., Li, A.Y., Perez De Leon, A.A. 2013. Acetylcholinesterase of the sand fly Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression and biochemical properties. Parasites & Vectors. 6(1):article 31.

Last Modified: 11/27/2014
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